Bi-directional Dual-flow-RootChip for Physiological Analysis of Plant Primary Roots Under Asymmetric Perfusion of Stress Treatments.

Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress-signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual-flow-RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as Arabidopsis thaliana, Nicotiana benthamiana, and Solanum lycopersicum, on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved. Key features Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments. Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550 µm width, and 500 µm height. Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines. Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC. Graphical overview Graphical overview of bi-dfRC fabrication, plantlet culture, and setup for root physiological analysis.(a) Schematic diagram depicting photolithography and replica molding, to produce a PDMS device. (b) Schematic diagram depicting seed culture off chip, followed by sub-culture of 4-day-old plantlets on chip. (c) Schematic diagram depicting microscopy and imaging setup, equipped with a media delivery system for asymmetric treatment introduction into the bi-dfRC microchannel root physiological analysis under varying conditions.

How plants solubilise seed fats: revisiting oleosin structure and function to inform commercial applications.

Plants store triacylglycerides in organelles called oil bodies, which are important fuel sources for germination. Oil bodies consist of a lipid core surrounded by an interfacial single layer membrane of phospholipids and proteins. Oleosins are highly conserved plant proteins that are important for oil body formation, solubilising the triacylglycerides, stabilising oil bodies, and playing a role in mobilising the fuel during the germination process. The domain structure of oleosins is well established, with N- and C-terminal domains that are hydrophilic flanking a long hydrophobic domain that is proposed to protrude into the triacylglyceride core of the oil body. However, beyond this general understanding, little molecular level detail on the structure is available and what is known is disputed. This lack of knowledge limits our understanding of oleosin function and concomitantly our ability to engineer them. Here, we review the state of play in the literature regarding oleosin structure and function, and provide some examples of how oleosins can be used in commercial settings.

Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation.

Environmental cues profoundly modulate cell proliferation and cell elongation to inform and direct plant growth and development. External phosphate (Pi) limitation inhibits primary root growth in many plant species. However, the underlying Pi sensory mechanisms are unknown. Here we genetically uncouple two Pi sensing pathways in the root apex of Arabidopsis thaliana. First, the rapid inhibition of cell elongation in the transition zone is controlled by transcription factor STOP1, by its direct target, ALMT1, encoding a malate channel, and by ferroxidase LPR1, which together mediate Fe and peroxidase-dependent cell wall stiffening. Second, during the subsequent slow inhibition of cell proliferation in the apical meristem, which is mediated by LPR1-dependent, but largely STOP1-ALMT1-independent, Fe and callose accumulate in the stem cell niche, leading to meristem reduction. Our work uncovers STOP1 and ALMT1 as a signalling pathway of low Pi availability and exuded malate as an unexpected apoplastic inhibitor of root cell wall expansion.

Long-term iron deficiency: Tracing changes in the proteome of different pea (Pisum sativum L.) cultivars.

UNLABELLED: Iron deficiency (-Fe) is one of the major problems in crop production. Dicots, like pea (Pisum sativum L.), are Strategy I plants, which induce a group of specific enzymes such as Fe(III)-chelate reductase (FRO), Fe responsive transporter (IRT) and H(+)-ATPase (HA) at the root plasma membrane under -Fe. Different species and cultivars have been shown to react diversely to -Fe. Furthermore, different kinds of experimental set-ups for -Fe have to be distinguished: i) short-term vs. long-term, ii) constant vs. acute alteration and iii) buffered vs. unbuffered systems. The presented work compares the effects of constant long-term -Fe in an unbuffered system on roots of four different pea cultivars in a timely manner (12, 19 and 25days). To differentiate the effects of -Fe and plant development, control plants (+Fe) were analyzed in comparison to -Fe plants. Besides physiological measurements, an integrative study was conducted using a comprehensive proteome analysis. Proteins, related to stress adaptation (e.g. HSP), reactive oxygen species related proteins and proteins of the mitochondrial electron transport were identified to be changed in their abundance. Regulations and possible functions of identified proteins are discussed. SIGNIFICANCE: Pea (Pisum sativum L.) belongs to the legume family (Fabaceae) and is an important crop plant due to high Fe, starch and protein contents. According to FAOSTAT data (September 2015), world production of the garden pea quadrupled from 1970 to 2012. Since the initial studies by Gregor Mendel, the garden pea became the most-characterized legume and has been used in numerous investigations in plant biochemistry and physiology, but is not well represented in the "omics"-related fields. A major limitation in pea production is the Fe availability from soils. Adaption mechanisms to Fe deficiency vary between species, and even cultivars have been shown to react diversely. A label-free proteomic approach, in combination with physiological measurements, was chosen to observe four different pea cultivars for 5 to 25days. Physiological and proteome data showed that cultivar Blauwschokker and Vroege were more susceptible to -Fe than cultivar Kelvedon (highly efficient) and GftR (semi-efficient). Proteomic data hint that the adaptation process to long-term -Fe takes place between days 19 and 25. Results show that adaptation processes of efficient cultivars are able to postpone secondary negative effects of long-term -Fe, possibly by stabilizing the protein metabolic processing and the mitochondrial electron transport components. This maintains the cellular energy proliferation, keeps ROS production low and postpones the mitochondrial cell death signal.

IPG-strips versus off-gel fractionation: advantages and limits of two-dimensional PAGE in separation of microsomal fractions of frequently used plant species and tissues.

The crucial cellular role of membrane proteins is generally known for all life forms. Depending on the species, tissue, compartment, function and physiological condition, membranes differ in their protein and lipid profiles. Additionally, occurrence of microdomains hampers quantitative protein solubilisation and therefore membrane proteomics remain a major challenge. In the present study sample preparation (TCA/acetone and methanol/chloroform precipitation with and without SDS pre-solubilisation) for two-dimensional PAGE were compared for microsomal fractions of leaves (Arabidopsis thaliana, Nicotiana tabaccum, Pisum sativum) and roots (P. sativum, Zea mays). Generally, pre-solubilisation with SDS impaired the resolution of the gels. All samples showed higher spot yields with TCA/acetone precipitation. Finally, we compared the results of conventional 2D-PAGE (IPG/SDS-PAGE) and the combination of off-gel fractionation in the first-dimension, 10% urea-SDS-PAGE in the second-dimension. Results showed that more spots are present in the alkaline pH range after off-gel fractionation then on conventional 2D-PAGE. For the first time, off-gel fractionation was combined with SDS/SDS-PAGE and BAC/SDS-PAGE to improve the resolution after off-gel fractionation. Transmembrane domains and GRAVY were calculated for all significantly identified spots resulting from the MALDI-TOF-TOF mass spectrometry showing that in the second dimension after off-gel fractionation 10.3% more transmembrane proteins were identified compared to IPG/SDS-PAGE.

Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan.

Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed.